E1-Mediated Recruitment of a UAF1-USP Deubiquitinase Complex Facilitates Human Papillomavirus DNA Replication

Abstract

The human papillomavirus (HPV) E1 helicase promotes viral DNA replication through its DNA unwinding activity and association with host factors. The E1 proteins from anogenital HPV types interact with the cellular WD repeat-containing factor UAF1 (formerly known as p80). Specific amino acid substitutions in E1 that impair this interaction inhibit maintenance of the viral episome in immortalized keratinocytes and reduce viral DNA replication by up to 70% in transient assays. In this study, we determined by affinity purification of UAF1 that it interacts with three deubiquitinating enzymes in C33A cervical carcinoma cells: USP1, a nuclear protein, and the two cytoplasmic enzymes USP12 and USP46. Coimmunoprecipitation experiments indicated that E1 assembles into a ternary complex with UAF1 and any one of these three USPs. Moreover, expression of E1 leads to a redistribution of USP12 and USP46 from the cytoplasm to the nucleus. Chromatin immunoprecipitation studies further revealed that E1 recruits these threes USPs to the viral origin in association with UAF1. The function of USP1, USP12, and USP46 in viral DNA replication was investigated by overproduction of catalytically inactive versions of these enzymes in transient assays. All three dominant negative USPs reduced HPV31 DNA replication by up to 60%, an effect that was specific, as it was not observed in assays performed with a truncated E1 lacking the UAF1-binding domain or with bovine papillomavirus 1 E1, which does not bind UAF1. These results highlight the importance of the USP1, USP12, and USP46 deubiquitinating enzymes in anogenital HPV DNA replication. IMPORTANCEHuman papillomaviruses are small DNA tumor viruses that induce benign and malignant lesions of the skin and mucosa. HPV types that infect the anogenital tract are the etiological agents of cervical cancer, the majority of anal cancers, and a growing proportion of head-and-neck cancers. Replication of the HPV genome requires the viral protein E1, a DNA helicase that also interacts with host factors to promote viral DNA synthesis. We previously reported that the E1 helicase from anogenital HPV types associates with the WD40 repeat-containing protein UAF1. Here, we show that UAF1 bridges the interaction of E1 with three deubiquitinating enzymes, USP1, USP12, and USP46. We further show that these deubiquitinases are recruited by E1/UAF1 to the viral origin of DNA replication and that overexpression of catalytically inactive versions of these enzymes reduces viral DNA replication. These results highlight the need for an E1-associated deubiquitinase activity in anogenital HPV genome replication.

Artificial Recruitment of UAF1-USP Complexes by a PHLPP1-E1 Chimeric Helicase Enhances Human Papillomavirus DNA Replication

“…To test if the stimulatory effect of PUBS described above was dependent on the enzymatic activity of UAF1-containing deubiquitinase complexes, we tested if it could be abrogated by overexpression of catalytically inactive USP1 (USP1ci), USP12ci, and USP46ci. We previously reported that these USPci enzymes inhibit the ability of wild-type E1 to support viral DNA replication by 40 to 70%, while having much less of an effect on the E1⌬ protein (13). The results presented in Fig.…”

“…This HA vector was constructed by substituting the 3F epitope of pCMV3Tag-1a (Stratagene) by an HA epitope. Expression vectors for RFP-USP1, USP12, and USP46 and their catalytically inactive versions were described previously (13). The plasmid used to express the HPV31 E1 UAF1-binding site (UBS) fused to YFP was previously described (referred to as N40-YFP in reference 8).…”

“…It was previously reported that the cellular PHLPP1 phosphatase interacts with UAF1 and is a substrate of either USP1, USP12, or USP46, depending on the study (17)(18)(19)(20)(21). To confirm that PHLPP1 interacts with UAF1, coimmunoprecipitation experiments were performed in transfected C33A cells transiently expressing UAF1 fused to a triple-Flag epitope (3F-UAF1) and PHLPP1 tagged with GFP (PHLPP1-GFP), essentially as described previously (13). These experiments confirmed that PHLPP1, and its close homolog PHLPP2, do interact with UAF1 in vivo (Fig.…”

“…In noninfected cells, UAF1 forms separate complexes with the three deubiquitinating enzymes USP1, USP12, and USP46 and stimulates their activity (9)(10)(11)(12). Accordingly, we recently determined that E1 assembles with UAF1 and either USP1, USP12, or USP46 into ternary complexes that are recruited to the origin of replication and whose deubiquitinase activity stimulates viral DNA replication in transient assays (13).…”

“…We selected to characterize the UBS from the cellular pleckstrin homology domain and leucine-rich repeat protein phosphatase 1 (PHLPP1), because this protein was found to interact with UAF1 in previous proteomic studies (including our own [13]) (data not shown) and to be a substrate of either USP1, USP12, or USP46, depending on the report (17)(18)(19)(20)(21). PHLPP1, and its close homologue PHLPP2, have been shown to regulate the proliferation and survival of normal and cancer cells through their ability to dephosphorylate a common set of substrates, which include the kinases Akt and PKC (22,23).…”